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Expression of the Pycnoporus cinnabarinus laccase gene in Aspergillus niger and characterization of the recombinant enzyme

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Author: Record, E. · Punt, P.J. · Chamkha, M. · Labat, M. · Hondel, C.A.M.J.J. van den · Asther, M.
Institution: Centraal Instituut voor Voedingsonderzoek TNO
Source:European Journal of Biochemistry, 2, 269, 602-609
Identifier: 72516
doi: doi:10.1046/j.0014-2956.2001.02690.x
Keywords: Nutrition · Amino Acid Sequence · Base Sequence · Basidiomycota · Blotting, Northern · Blotting, Western · Cloning, Molecular · DNA, Complementary · Hydrogen-Ion Concentration · Isoelectric Point · Kinetics · Laccase · Molecular Weight · Oxidoreductases · Recombinant Proteins · Temperature · Aspergillus · Aspergillus niger · Prokaryota · Pycnoporus cinnabarinus


Pycnoporus cinnabarinus laccase lac1 gene was overexpressed in Aspergillus niger, a well-known fungal host producing a large amount of homologous or heterologous enzymes for industrial applications. The corresponding cDNA was placed under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter as a strong and constitutive promoter. The laccase signal peptide or the glucoamylase preprosequence of A. niger was used to target the secretion. Both signal peptides directed the secretion of laccase into the culture medium as an active protein, but the A. niger preprosequence allowed an 80-fold increase in laccase production. The identity of the recombinant protein was further confirmed by immunodetection using Western blot analysis and N-terminal sequencing. The molecular mass of the mature laccase was 70 kDa as expected, similar to that of the native form, suggesting no hyperglycosylation. The recombinant laccase was purified in a three-step procedure including a fractionated precipitation using ammonium sulfate, and a concentration by ultrafiltration followed by a Mono Q column. All the characteristics of the recombinant laccase are in agreement with those of the native laccase. This is the first report of the production of a white-rot laccase in A. niger. Molecular Sequence Numbers: GENBANK: AF170093; Chemicals/CAS: DNA, Complementary; Laccase, EC; Oxidoreductases, EC 1.-; Recombinant Proteins